The present invention relates to a new bacterial strain of the genus Vibrio, to the exopolysaccharides produced by the said strain and to their uses.
Some microorganisms obtained from the deep submarine hydrothermal medium produce biomolecules whose particular structure and composition confer on them properties of great potential industrial interest; among these biomolecules are a wide variety of polysaccharides some of which have already been the subject of studies intended to determine their structures and their properties.
The studies mentioned below have related more specifically to the exopolysaccharides (EPS) excreted by bacteria of the genus Alteromonas which are cultured under laboratory conditions, and in particular on glucose-enriched medium.
VINCENT et al. [Appl. Environ. Microbiol., 60, 4134-4141 (1994)] have thus described an exopolysaccharide, called EPS-1545, excreted by a strain, designated by the reference HYD-1545, of a bacteria of the genus Alteromonas. The EPS obtained by ethanol precipitation comprises between 49 and 55% of neutral monosaccharides, and between 32.5 and 39% of uronic acids.
GUEZENNEC et al., [Carbohydrate Polymers, 24, 287-294 (1994)] have classified the EPSs produced by various bacteria of the genus Alteromonas into 5 different groups on the basis of their composition: group 1 consists of EPS comprising between about 50 and 60% of neutral monosaccharides, about 10% of uronic acids, between 0.5 and 3.7% of osamines, and between 11.5 and 21.5% of sulphates; group 2 consists of EPS comprising about 50% of neutral monosaccharides, possessing a low content of uronic acids (8%), comprising between 1 and 3.2% of osamines, and whose content of sulphates is between less than 10% and 13%; the group 3 EPSs comprise between 40 and 50% of neutral monosaccharides, have a very low content of uronic acids (between S and 7%), comprise between 1.2 and 1.7% of osamines, and have a content of sulphates varying between 8.9 and 17.2; group 4 consists of EPS comprising 46 to 49% of neutral monosaccharides, possessing a high content of uronic acids (between 34 and 40%), comprising between 0.2 and 1.6% of osamines, and having a content of sulphates of between 9.7 and 13%; group 5 consists of EPS having a relatively low content of neutral monosaccharides (38 to 47%), a high content of uronic acids (26 to 32%), comprising between 1 and 1.6% of osamines, and between 5.2 and 10.1% of sulphates; one of the EPSs of this group comprises, in addition, one hexuronic acid carrying a lactate substituent.
A number of the strains and polymers described in the publications by VINCENT et al. and GUEZENNEC et al. cited above are the subject of application PCT FR 94/00169 published under WO 94/18340.
Recently, RAGUENES et al. [Appl. Environ. Microbiol, 62, 67-73 (1996)] have described an EPS, different from the preceding ones, which is obtained from a bacterium of the genus Alteromonas (Alteromonas macleodii subsp. fijiensis). This EPS comprises about 38% of neutral monosaccharides, 38% of uronic acids, 2.3% of osamines and about 5% of sulphates.
The inventors, continuing their research studies on bacteria obtained from the deep submarine hydrothermal medium have now isolated from Pompei worm (Alvinella pompejana) a new strain, called HE800, belonging to the genus Vibrio. This strain, which was deposited on Oct. 17, 1995 at the CNCM (Collection Nationale de Cultures de Microorganismes, 28, rue du Docteur Roux, 75724 PARIS, FRANCE) under number I-1629, represents a new species of Vibrio, for which the name Vibrio diabolicus is proposed.
Vibrio diabolicus is a Gram-negative bacillus, about 0.8 xcexcwide and 2.2 xcexcm long; it is mobile with the aid of a polar flagellum in liquid medium and of peritrichous flagella in solid medium; it is a non-encapsulated, non-pigmented and non-luminescent bacterium.
It is a catalase+, cytochrome oxidase+, chitinase+, faculative anaerobic bacterium. It reduces nitrates to nitrites. It is sensitive to the vibriostatic agent 0/129 (2, 4-diamino-6, 7-diisopropylpteridine).
It uses a wide variety of carbon substrates; it can use in particular, as sole carbon source, any one of the following substrates: glycerol, ribose, galactose, glucose, fructose, mannose, mannitol, N-acetylglucosamine, maltose, sucrose, trehalose, starch, glycogen, gluconate, caprate, citrate and malate.
Its growth is optimum at a temperature of between 30 and 45xc2x0 C., a pH of between 7 and 8, and a salinity of between 20 and 50 g/l of NaCl; its generation time under these conditions is between 18 and 28 minutes.
The G+C content of its DNA is 49.6%. The phylogenic analysis of the 16S rRNA gene (according to the protocol established by RUIMY et al. [Int. J. Syst. Bacteriol 44, 416-426 (1994)] have made it possible to establish that it belongs to a well-defined taxon which also includes Vibrio mytili, Vibrio nereis and Vibrio tubiashii. The results of this analysis are illustrated by FIG. 1. The percentage homology of the DNA of the HE800 strain with that of the 3 most closely related Vibrio mentioned above is 27%, 15% and 5%, respectively.
Table I below illustrates the metabolic properties of the HE800 strain (only the positive responses are included in this table)
The above morphological, biochemical and phylogenetic characteristics make it possible to include the HE800 strain in the genus Vibrio [BAUMANN et al.: Genus Vibrio, Bergey""s Manual of Systematic Bacteriology, Vol. 1, 342-352. (1984) KRIEG, and HOIT (ed.); The Williams and Wilkins Co., Baltimore].
Because the DNA/DNA percentage homology with the three subspecies of the abovementioned genus Vibrio is less than 30%, the HE800 strain may be considered as representing a new species of Vibrio [WAYNE et al., Report of the Ad Hoc Committee on reconciliation of approaches to bacterial systematics, Int. J. Syst. Bacteriol. 463-464, (1987)], for which the name: Vibrio diabolicus is proposed.
The subject of the present invention is strains of bacteria possessing the above-defined characteristics of the species Vibrio diabolicus, and in particular the HE800 strain; this includes in particular bacteria whose DNA has a percentage homology greater than 28%, preferably greater than 30%, and advantageously greater than 50%, with the DNA of the HE800 strain. This subject also includes the bacteria obtained from bacteria of the species Vibrio diabolicus, and in particular of the strain HE800, by mutation, or by genetic recombination, such as for example bacteria of the species Vibrio diabolicus harbouring a plasmid carrying a heterologous gene.
The present invention also includes the various products which may be obtained from the said bacteria, which comprises in particular their cellular fractions, the enzymes as well as the nucleic acid preparations which may be extracted therefrom, as well as the products excreted or secreted by these bacteria.
This comprises polysaccharides capable of being obtained from Vibrio diabolicus culture supernatants, and in particular an exopolysaccharide capable of being obtained by ethanol precipitation, from culture supernatants of the HE800 strain.
The analysis carried out by the inventors of an exopolysaccharide in accordance with the present invention, produced by the HE800 strain, reveals the following characteristics, determined from a preparation of the said polysaccharide comprising about 1% by weight of proteins:
it does not comprise neutral monosaccharides;
its content of osamines is about 30xc2x15% by weight;
its content of uronic acids is about 32xc2x15% by weight;
its monosaccharide composition, determined after acid methanolysis, is the following: about 11.2% by weight of glucuronic acid, about 18% by weight of N-acetylglucosamine, about 7.9% by weight of N-acetylgalactosamine, that is to say 1.4 mol of N-acetylglucosamine and 0.6 mol of N-acetylgalactosamine per 1 mol of glucuronic acid;
it does not comprise sulphated saccharide units;
its intrinsic viscosity is of the order of 570 ml/g;
its mean molecular weight is of the order of 800,000 Da.
This polysaccharide possesses a composition similar to that of heparin, which is commonly used in the pharmaceutical industry, in particular for its anticoagulant and antithrombotic properties. It however differs therefrom in particular by the presence of N-acetylgalactosamine and by the absence of sulphate groups.
The subject of the present invention is also fractions of the polysaccharide defined above, and in particular low-molecular weight, and highly sulphated, fractions.
Because of its structure, this polysaccharide can be used in particular in the context of the pharmaceutical industry, for example as raw material in the context of the production of medicaments. For this purpose, it may for example be modified, in particular by the chemical or enzymatic route in order to obtain fractions which are sulphated, and:or of low molecular weight, which can be used for example as antiviral, antitumour or antithrombotic agents.
The present invention will be understood more clearly with the aid of the additional description which follows, which refers to examples describing the preparation and the characterization of polysaccharides in accordance with the invention.
It goes without saying, however, that these examples are given solely by way of illustration of the subject of the invention and do not in any manner constitute a limitation thereto.